美国康奈尔大学Elizabeth H. Kellogg团队解析出全CRISPR RNA引导的转座子整合复合物结构。相关论文于2022年11月28日在线发表在《自然》杂志上。

研究人员表示，CRISPR相关转座子（CAST）是可编程的移动遗传元件，利用RNA引导的机制插入大型DNA货物。CAST元件包含多个保守基因，包括：一个CRISPR效应器（Cas12k或Cascade），一个AAA+调节器（TnsC），一个转座酶（TnsA/TnsB），以及一个靶点相关因子（TniQ）。这些成分被认为是通过形成一个多亚单位的转座整合复合物（转座体）来合作整合DNA。

研究人员重组了来自Scytonema hofmannii的大约1MDa的V-K型CAST（ShCAST）转座体，并使用单粒子冷冻电镜确定了近原子分辨率（3.5Å）的结构。转座体结构揭示了各组成部分之间的模块化关联。Cas12k与核糖体亚单位S15和TniQ形成一个复合体，稳定了全R环的形成。TnsC与TniQ和TnsB有专门的交互界面。有趣的是，研究人员在TnsC的C端面观察到新的TnsC-TnsB相互作用，这有助于刺激ATP酶的活性。尽管TnsC的低聚物组装稍微偏离了孤立的螺旋结构，但TnsC结合的靶标DNA构象在转座体中却有很大的不同。因此，TnsC在整个转座体中进行新的蛋白质-DNA相互作用，这对转座活动非常重要。

最后，研究人员发现了两个不同的转座体群体，它们在TniQ附近的DNA接触上有所不同。这表明，与CRISPR效应器的联系可以是灵活的。这种ShCAST转座体结构大大增强了人们对CAST转座系统的理解，并提出了改进CAST转座的途径，以便进行精确的基因组编辑应用。

附：英文原文

Title: Structures of the holo CRISPR RNA-guided transposon integration complex

Author: Park, Jung-Un, Tsai, Amy Wei-Lun, Rizo, Alexandrea N., Truong, Vinh H., Wellner, Tristan X., Schargel, Richard D., Kellogg, Elizabeth H.

Issue&Volume: 2022-11-28

Abstract: CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargo using an RNA-guided mechanism1-3. CAST elements contain multiple conserved genes, including: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA/TnsB), and a target site associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstitute the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined the structure to near-atomic resolution (3.5 ) using single particle cryo-EM. Transpososome architecture reveals modular association between components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Interestingly, we observe novel TnsC-TnsB interactions at the C-terminal face of TnsC, which contribute to stimulation of ATPase activity. Although the TnsC oligomeric assembly slightly deviates from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs dramatically in the transpososome. As a consequence, TnsC makes new protein-DNA interactions throughout the transpososome that are important for transposition activity. Finally, we discover two distinct transpososome populations which differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure significantly enhances our understanding of CAST transposition systems and suggests avenues for improving CAST transposition for precision genome-editing applications.

DOI: 10.1038/s41586-022-05573-5

Source: https://www.nature.com/articles/s41586-022-05573-5